Expression of recombinant neutralizing anti-HIV-1 antibodies in bacteria and eukaryotic cells
Author: Yari, Fayezeh
Date: 2007-01-25
Location: Föreläsningssalen CMM, plan 00, Karolinska Universitetssjukhuset, Solna
Time: 13.00
Department: Institutionen för medicin / Department of Medicine
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Thesis (2.773Mb)
Abstract
The main purpose of this study was to investigate and optimize techniques for eukaryotic expression of antibody molecules (IgG, Fab and scFV-ê formats), derived from an immune phage displayed Fab library and reactive against HIV-1 glycoproteins gp120 and gp41.
Light and heavy-chain (Fd) DNA from a number of Fab clones were transferred from a prokaryotic phagemid vectors to mammalian expression vectors. COS-7 cells were used for transient expression of Fab-fragments. In addition, by cloning cDNA encoding the Fc fragment downstream of Fd, full length, human IgG1 plasmids were created and transfected into Chinese Hamster Ovary (CHO) cells. Fab fragments of certain clones showed increased virus neutralizing activity when expressed in CHO cells, compared to when expressed in Escherichia coli. Neutralizing capacity of Fab and IgG expressed in mammalian cells were almost the same, indicating that bivalency did not confer improved neutralization efficacy.
Next, conditional expression systems for IgG in mammalian cells were constructed based on the tetracycline operon. HeLa and CHO cell lines that produced prtTA/prtTA-M2s (reverse tetresponsive transcriptional activator) were established. The cDNA for Fab fragments to tetanus toxioid, HIV-1 gp41 and gpl20 were transformed into a single chain format, scFV-ê (VH-linker-VL+CL), cloned downstream of the tetracycline responsive element, and transfected into the HeLa-tet on and CHO-tet on cell lines. The gene expression in presence of doxycycline was increased 10 -100 fold; a very low background level was seen when the tetracycline substrate was withdrawn.
To improve yields of secreted proteins, a series of vectors adapted for inducible expression of IgG1 in Drosophila S2 cells were constructed. High expression levels were obtained in stable cell lines: from 0.5-1 mg/l in transient expression, but up to 35mg/l from stable cell lines when accumulated during 10 days. Binding characteristics for IgG1 expressed in S2 cells were indistinguishable of the same clone expressed in mammalian cells, as assessed for anti-HIV and anti-HCV antibodies.
Finally, human Fab antibodies against a putative neutralizing epitope in gp41 of HIV-1 were isolated using overlapping synthetic peptides representing the epitope. The heavy chains were clonally unrelated, despite that almost all clones showed strong specificity to one of the three peptides. The results from E.coli produced Fab indicated that certain clones could neutralize up to 4 of 5 primary HIV-1 isolates. Improved neutralization capacities were found when Fab and/or IgG were expressed in COS, CHO and Drosophila S2 cells. Immunoprecipitation by S2- produced IgG of gp41 and gpl60 containing cell lysates, confirmed the binding to the cognate antigen.
In conclusion, human antibodies isolated as Fab fragments could be expressed in eukaryotic cells as Fab or IgG. Several of our clones were improved in their antiviral activities by the expression in mammalian or insect cells, compared to when the same clone was produced in E. coli. The antiHIV1 antibodies had promising in vitro HIV-1 neutralizing effects and may be further explored as molecular tools for elucidating detailed mechanisms of virus neutralization via the epitopes involved, which may also guide vaccine design. Potent human antiviral antibodies may become useful as prophylaxis or after accidental exposure to the virus.
Light and heavy-chain (Fd) DNA from a number of Fab clones were transferred from a prokaryotic phagemid vectors to mammalian expression vectors. COS-7 cells were used for transient expression of Fab-fragments. In addition, by cloning cDNA encoding the Fc fragment downstream of Fd, full length, human IgG1 plasmids were created and transfected into Chinese Hamster Ovary (CHO) cells. Fab fragments of certain clones showed increased virus neutralizing activity when expressed in CHO cells, compared to when expressed in Escherichia coli. Neutralizing capacity of Fab and IgG expressed in mammalian cells were almost the same, indicating that bivalency did not confer improved neutralization efficacy.
Next, conditional expression systems for IgG in mammalian cells were constructed based on the tetracycline operon. HeLa and CHO cell lines that produced prtTA/prtTA-M2s (reverse tetresponsive transcriptional activator) were established. The cDNA for Fab fragments to tetanus toxioid, HIV-1 gp41 and gpl20 were transformed into a single chain format, scFV-ê (VH-linker-VL+CL), cloned downstream of the tetracycline responsive element, and transfected into the HeLa-tet on and CHO-tet on cell lines. The gene expression in presence of doxycycline was increased 10 -100 fold; a very low background level was seen when the tetracycline substrate was withdrawn.
To improve yields of secreted proteins, a series of vectors adapted for inducible expression of IgG1 in Drosophila S2 cells were constructed. High expression levels were obtained in stable cell lines: from 0.5-1 mg/l in transient expression, but up to 35mg/l from stable cell lines when accumulated during 10 days. Binding characteristics for IgG1 expressed in S2 cells were indistinguishable of the same clone expressed in mammalian cells, as assessed for anti-HIV and anti-HCV antibodies.
Finally, human Fab antibodies against a putative neutralizing epitope in gp41 of HIV-1 were isolated using overlapping synthetic peptides representing the epitope. The heavy chains were clonally unrelated, despite that almost all clones showed strong specificity to one of the three peptides. The results from E.coli produced Fab indicated that certain clones could neutralize up to 4 of 5 primary HIV-1 isolates. Improved neutralization capacities were found when Fab and/or IgG were expressed in COS, CHO and Drosophila S2 cells. Immunoprecipitation by S2- produced IgG of gp41 and gpl60 containing cell lysates, confirmed the binding to the cognate antigen.
In conclusion, human antibodies isolated as Fab fragments could be expressed in eukaryotic cells as Fab or IgG. Several of our clones were improved in their antiviral activities by the expression in mammalian or insect cells, compared to when the same clone was produced in E. coli. The antiHIV1 antibodies had promising in vitro HIV-1 neutralizing effects and may be further explored as molecular tools for elucidating detailed mechanisms of virus neutralization via the epitopes involved, which may also guide vaccine design. Potent human antiviral antibodies may become useful as prophylaxis or after accidental exposure to the virus.
List of papers:
I. Samuelsson A, Yari F, Hinkula J, Ersoy O, Norrby E, Persson MA. (1996). Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells. Eur J Immunol. 26(12): 3029-34.
Pubmed
View record in Web of Science®
II. Yari F, Persson M.A.A. (2006). Tetracycline regulated scFv-ê expression in eukaryotic cells. [Manuscript]
III. Johansson DX, Drakenberg K, Hopmann KH, Schmidt A, Yari F, Hinkula J, Persson MA. (2006). Efficient expression of recombinant human monoclonal antibodies in Drosophila S2 cells. J Immunol Methods. [Accepted]
Pubmed
View record in Web of Science®
IV. Hinkula J, Yari F, Lundin K, Bratt G, Holmberg V, Sandström E, Wahren B, Persson M.A.A (2006). Human anti gp41 monoclonal antibodies selected against a putative neutralizing epitope inhibit infectivity of primary isolates. [Manuscript]
I. Samuelsson A, Yari F, Hinkula J, Ersoy O, Norrby E, Persson MA. (1996). Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells. Eur J Immunol. 26(12): 3029-34.
Pubmed
View record in Web of Science®
II. Yari F, Persson M.A.A. (2006). Tetracycline regulated scFv-ê expression in eukaryotic cells. [Manuscript]
III. Johansson DX, Drakenberg K, Hopmann KH, Schmidt A, Yari F, Hinkula J, Persson MA. (2006). Efficient expression of recombinant human monoclonal antibodies in Drosophila S2 cells. J Immunol Methods. [Accepted]
Pubmed
View record in Web of Science®
IV. Hinkula J, Yari F, Lundin K, Bratt G, Holmberg V, Sandström E, Wahren B, Persson M.A.A (2006). Human anti gp41 monoclonal antibodies selected against a putative neutralizing epitope inhibit infectivity of primary isolates. [Manuscript]
Issue date: 2007-01-04
Rights:
Publication year: 2007
ISBN: 978-91-7357-079-4
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